Nucleic Acids Research Advance Access published online on January 21, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1172
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Structural Biology |
The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site
1Department of Biochemistry, 2X-ray Crystallography Core Laboratory, The University of Texas Health Science Center San Antonio, San Antonio, TX-78229, USA, 3Excellence Center: Macromolecular Complexes and Institut für Molekulare Biowissenschaften, Johann-Wolfgang-Goethe-Universität, 60438 Frankfurt/M., Germany, 4Center for Analytical Ultracentrifugation of Macromolecular Assemblies, The University of Texas Health Science Center San Antonio and 5Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, Texas 78229, USA
*To whom correspondence should be addressed. Tel: ++1 210 567 8781; Fax: ++1 210 567 6595; Email: jewoe{at}biochem.uthscsa.edu Correspondence may also be addressed to Karl-Dieter Entian. Tel: +49 69 798 29525; Fax: +49 69 798 29527; Email: entian{at}bio.uni-frankfurt.de
Received October 23, 2007. Revised December 18, 2007. Accepted December 20, 2007.
Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1ts) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 Å resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 Å resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 Å resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.